Inference of subclonal architecture of tumors across multiple timepoints in the paired longitudinal (PL) cohort

Background

This notebook is for preparing input files for pyclone-vi. The same files with a minor modification will be used as an input for FastClone.

Set up

suppressPackageStartupMessages({
  library(tidyverse)
  library(readxl)
  #library(cDriver) # Calculate CCF, https://github.com/hanasusak/cDriver/
})

Directories and File Inputs/Outputs

# Detect the ".git" folder -- this will be in the project root directory
# Use this as the root directory to ensure proper sourcing of functions 
# no matter where this is called from
root_dir <- rprojroot::find_root(rprojroot::has_dir(".git"))
analysis_dir <- file.path(root_dir, "analyses", "tumor-clone-inference")
input_dir <- file.path(analysis_dir, "input")
data_dir <- file.path(root_dir, "data")
files_dir <- file.path(root_dir, "analyses", "sample-distribution-analysis", "results")
maf_files_dir <- file.path(root_dir, "analyses", "tmb-vaf-longitudinal", "results")
#tmb_input_dir <- file.path(root_dir, "analyses", "tmb-vaf-longitudinal", "input")
#scratch_dir <- file.path(root_dir, "scratch")

# Input files
pbta_file <- file.path(files_dir, "pbta.tsv") # file from add-sample-distribution module
maf_file <- file.path(data_dir, "snv-consensus-plus-hotspots.maf.tsv.gz")
tmb_file <- file.path(maf_files_dir, "tmb_vaf_genomic.tsv")
genomic_paired_file <- file.path(files_dir, "genomic_assays_matched_time_points.tsv") # file from add-sample-distribution module
nautilus_dec_file <- file.path(input_dir, "deceased_samples.xlsx") 
palette_file <- file.path(root_dir, "figures", "palettes", "tumor_descriptor_color_palette.tsv")
#tmb_all_file <- file.path(tmb_input_dir, "snv-mutation-tmb-coding.tsv")


# EXAMPLE 
# But to replace with dir with cns data inferred by CNVkit
# PT_Z4BF2NSB_dir <- file.path(input_dir, "cnvkit_data_example/PT_Z4BF2NSB") 
cns_dir <- file.path(input_dir, "cnvkit_data") 

# File path to results directory
results_dir <-
  file.path(analysis_dir, "results")
if (!dir.exists(results_dir)) {
  dir.create(results_dir)
}

# File path to input directory
pyclonevi_input_dir <-
  file.path(analysis_dir, "results", "pyclone-vi-input")
if (!dir.exists(pyclonevi_input_dir)) {
  dir.create(pyclonevi_input_dir)
}

# File path to input directory
fastclone_input_dir <-
  file.path(analysis_dir, "results", "fastclone-input")
if (!dir.exists(pyclonevi_input_dir)) {
  dir.create(pyclonevi_input_dir)
}


# File path to plot directory
pyclone_plots_dir <-
  file.path(analysis_dir, "plots", "pyclone-vi")
if (!dir.exists(pyclone_plots_dir)) {
  dir.create(pyclone_plots_dir)
}


source(paste0(root_dir, "/figures/scripts/theme.R"))

Load and process data

# Let's count #specimens per sample 
# We will remove any samples with less than 2 specimens as phylogenies require at least 3 taxa
kids_specimens_n_df <- genomic_paired_df %>% 
  select(Kids_First_Participant_ID, Kids_First_Biospecimen_ID, tumor_descriptor) %>% 
  unique() %>% 
  dplyr::count(Kids_First_Participant_ID) %>% 
  dplyr::mutate(kids_specimens_n = glue::glue("{Kids_First_Participant_ID}  (N={n})")) %>%
  dplyr::rename(kids_specimens_number = n) %>% 
  filter(!kids_specimens_number <= 2) %>%

  left_join(genomic_paired_df, by = c("Kids_First_Participant_ID")) %>%
  #left_join(maf_df) %>% 
  filter(!is.na(Chromosome)) 


# Let's confirm and add the number of timepoints per sample
# In case we lost any from filtering hypermutants and high reads/alteration
timepoints_n_df <- kids_specimens_n_df %>% 
  select(Kids_First_Participant_ID, tumor_descriptor) %>% 
  unique() %>% 
  dplyr::count(Kids_First_Participant_ID) %>% 
  dplyr::mutate(kids_timepoints_n = glue::glue("{Kids_First_Participant_ID}  (N={n})")) %>%
  dplyr::rename(kids_timepoints_number = n) %>% 
  filter(!kids_timepoints_number <= 1)

df <- timepoints_n_df %>% 
  left_join(kids_specimens_n_df, by = c("Kids_First_Participant_ID")) %>% 
  write_tsv(file.path(results_dir, "samples_eligible_for_phylogeny.tsv"))

# Number of samples in the `genomic_paired_df` with > 3 biospecimens and >2 timepoints
samples_with_3bs <- print(length(unique(df$Kids_First_Participant_ID)))

[1] 34

samples_with_3bs <- print(unique(df$Kids_First_Participant_ID))

 [1] "PT_04V47WFC" "PT_0DWRY9ZX" "PT_19GCSK2S" "PT_1H2REHT2" "PT_23NZGSRJ" "PT_3KM9W8S8" "PT_75HRTX4S" "PT_8GN3TQRM" "PT_962TCBVR" "PT_99S5BPE3" "PT_9S6WMQ92"
[12] "PT_AQWDQW27" "PT_C1RDBCVM" "PT_CWXSP19D" "PT_D6AJHDST" "PT_DVXE38EX" "PT_EQX0VT4F" "PT_GTHZF21E" "PT_HFQNKP5X" "PT_HHG37M6W" "PT_HJMP6PH2" "PT_K8ZV7APT"
[23] "PT_KBFM551M" "PT_KTRJ8TFY" "PT_KZ56XHJT" "PT_MDWPRDBT" "PT_MNSEJCDM" "PT_N8W26H19" "PT_NJQ26FHN" "PT_NK8A49X5" "PT_S4YNE17X" "PT_TP6GS00H" "PT_Z4BF2NSB"
[34] "PT_ZZRBX5JT"

  
# List with samples eligible for phylogeny
# I added the information about to use for phylogenetic inferences
list_df <- df %>% 
  select(Kids_First_Participant_ID) %>% 
  unique() %>% 
  mutate(somatic_germline_phylogeny = case_when(grepl("PT_KZ56XHJT|PT_KTRJ8TFY", Kids_First_Participant_ID) ~ "yes",
                                        TRUE ~ "not_yet")) %>% 
  write_tsv(file.path(results_dir, "samples_eligible_for_phylogeny_list.tsv"))

# Remove large files
rm(genomic_paired_df, kids_specimens_n_df, tmb_df)

Add Nautilus location for Deceased specimens

nautilus_dec_df <- read_excel(nautilus_dec_file) %>% 
  right_join(df, by = c("sample_id", "aliquot_id", "tumor_descriptor")) %>%
  dplyr::mutate(mutation_id = paste(Kids_First_Participant_ID, tumor_descriptor, Kids_First_Biospecimen_ID, Chromosome, Start_Position, Reference_Allele, Tumor_Seq_Allele2, sep = ":")) #%>% 
  #write_tsv(file.path(results_dir, "nautilus_dec.tsv"))

list_df <- nautilus_dec_df %>% 
  select(Kids_First_Participant_ID, Kids_First_Biospecimen_ID, `Note field from Nautilus of initial parent`) %>% 
  unique() %>% 
  write_tsv(file.path(results_dir, "nautilus_dec_list.tsv"))

Other, maybe to delete

# Make list with samples with >2 biospecimens at Deceased timepoint
dec_n_df <- df %>% 
  filter(tumor_descriptor == "Deceased") %>% 
  select(Kids_First_Participant_ID, tumor_descriptor, Kids_First_Biospecimen_ID) %>% 
  unique() %>% 
  dplyr::count(Kids_First_Participant_ID) %>% 
 # dplyr::mutate(kids_dec_n = glue::glue("{Kids_First_Participant_ID}  (N={n})")) %>%
  dplyr::rename(kids_deceased_bs_number = n) %>% 
  filter(!kids_deceased_bs_number <= 1) %>% 
  write_tsv(file.path(results_dir, "kids_dec_multiple_bs_list.tsv"))

###-----------------------------------------------------
# let's look into PT_3KM9W8S8
#PT_3KM9W8S8_df <- nautilus_dec_df %>% 
#  filter(Kids_First_Participant_ID == "PT_3KM9W8S8",
 #        Kids_First_Biospecimen_ID == "BS_2NQXY528") %>% 
#  select(Kids_First_Participant_ID, Kids_First_Biospecimen_ID, `Note field from Nautilus of initial parent`) %>% 
#  unique() %>% 
#  write_tsv(file.path(results_dir, "nautilus_dec_list.tsv"))
  
#bs_id <- print(unique(PT_3KM9W8S8_df$Kids_First_Biospecimen_ID))

Create pyclone input files

We need to generate the input files according to the method’s template. Phylogenetic methods require at least 2 samples per tumor site (multiregional sampling per anatomical site). Here, we will consider kids samples with more than 2 timepoints with one or more biospecimens. We will compare later differences in samples with single vs multiple biospecimens.

# Create pyclone df for all samples
pyclone_all_samples_df <- nautilus_dec_df %>%
  select(Kids_First_Participant_ID, Kids_First_Biospecimen_ID, cg_id, 
         cg_id_kids, mutation_id, sample_id,
         tumor_descriptor, Chromosome, Start_Position, 
         Reference_Allele, Tumor_Seq_Allele2, t_ref_count, t_alt_count,
         normal_cn, tumor_fraction, mutation_count) %>% 
  
  # change names to match input requirements
  dplyr::rename("ref_counts" = "t_ref_count", 
                "alt_counts" = "t_alt_count",
                "tumour_content" = "tumor_fraction") %>%
  select(Kids_First_Participant_ID, Kids_First_Biospecimen_ID, tumor_descriptor, 
         cg_id_kids, mutation_id, sample_id, ref_counts, 
         alt_counts, normal_cn, tumour_content, mutation_count)

samples_pyclone <- print(unique(pyclone_all_samples_df$Kids_First_Participant_ID))

 [1] "PT_04V47WFC" "PT_0DWRY9ZX" "PT_19GCSK2S" "PT_1H2REHT2" "PT_23NZGSRJ" "PT_3KM9W8S8" "PT_75HRTX4S" "PT_8GN3TQRM" "PT_962TCBVR" "PT_99S5BPE3" "PT_9S6WMQ92"
[12] "PT_AQWDQW27" "PT_C1RDBCVM" "PT_CWXSP19D" "PT_D6AJHDST" "PT_DVXE38EX" "PT_EQX0VT4F" "PT_GTHZF21E" "PT_HFQNKP5X" "PT_HHG37M6W" "PT_HJMP6PH2" "PT_K8ZV7APT"
[23] "PT_KBFM551M" "PT_KTRJ8TFY" "PT_KZ56XHJT" "PT_MDWPRDBT" "PT_MNSEJCDM" "PT_N8W26H19" "PT_NJQ26FHN" "PT_NK8A49X5" "PT_S4YNE17X" "PT_TP6GS00H" "PT_Z4BF2NSB"
[34] "PT_ZZRBX5JT"

# Remove large files
# rm(nautilus_dec_df)

# I will test one HGG dataset for now
# PT_Z4BF2NSB

#PT_Z4BF2NSB_DF <- pyclone_all_samples_df %>% 
#  filter(Kids_First_Participant_ID == "PT_Z4BF2NSB")
                   

Add major_cn, minor_cn columns from cns files

# Save df with all data together
# We will need this to prepare the input fiel for ClonEvol
pyclone_input_all <- nautilus_dec_df %>%
  select(mutation_id, VAF, CCF, gene_protein, Hugo_Symbol) %>% 
  right_join(pyclone_input) %>% 
  write_tsv(file.path(results_dir, "pyclone_input_all_data.tsv"))

Joining with `by = join_by(mutation_id, Hugo_Symbol)`

Save input files

for (i in 1:length(data_dir) ) { 
  
  # Create sample_name
  sample_name <-  unique(as.character(gsub(".call.cns", "", str_split_fixed(data_dir[i], "/", 13)[,11])))
  #sample_name <-  unique(as.character(gsub(".call.cns", "", str_split_fixed(data_dir[i], "/", 10)[,9])))
  sample_name <- sort(sample_name, decreasing = FALSE)
  print(sample_name)
  
for (x in seq_along(sample_name) ) { 
  
  # Save input file for pyclone-vi
  pyclone_fname <- paste0(pyclonevi_input_dir, "/", sample_name[x], ".tsv")
  print(pyclone_fname)
  
  pyclone_input_subset <- pyclone_input %>%
    filter(Kids_First_Participant_ID == sample_name[x]) %>% 
    #select(-c(Kids_First_Participant_ID)) %>% 
  write_tsv(file.path(pyclone_fname))
  
  
  # Save input file for FastClone
  fastclone_fname <- paste0(fastclone_input_dir, "/", sample_name[x], ".tsv")
  print(fastclone_fname)
  
  fastclone_input_subset <- pyclone_input %>%
    dplyr::rename("var_counts" = "alt_counts") %>% 
    filter(Kids_First_Participant_ID == sample_name[x]) %>% 
    #select(-c(Kids_First_Participant_ID)) %>% 
  write_tsv(file.path(fastclone_fname))
  
  }
}

[1] "PT_KTRJ8TFY"
[1] "/Users/chronia/CHOP/GitHub/pbta-tumor-evolution/analyses/tumor-clone-inference/results/pyclone-vi-input/PT_KTRJ8TFY.tsv"
[1] "/Users/chronia/CHOP/GitHub/pbta-tumor-evolution/analyses/tumor-clone-inference/results/fastclone-input/PT_KTRJ8TFY.tsv"
[1] "PT_KTRJ8TFY"
[1] "/Users/chronia/CHOP/GitHub/pbta-tumor-evolution/analyses/tumor-clone-inference/results/pyclone-vi-input/PT_KTRJ8TFY.tsv"
[1] "/Users/chronia/CHOP/GitHub/pbta-tumor-evolution/analyses/tumor-clone-inference/results/fastclone-input/PT_KTRJ8TFY.tsv"
[1] "PT_KTRJ8TFY"
[1] "/Users/chronia/CHOP/GitHub/pbta-tumor-evolution/analyses/tumor-clone-inference/results/pyclone-vi-input/PT_KTRJ8TFY.tsv"
[1] "/Users/chronia/CHOP/GitHub/pbta-tumor-evolution/analyses/tumor-clone-inference/results/fastclone-input/PT_KTRJ8TFY.tsv"
[1] "PT_KTRJ8TFY"
[1] "/Users/chronia/CHOP/GitHub/pbta-tumor-evolution/analyses/tumor-clone-inference/results/pyclone-vi-input/PT_KTRJ8TFY.tsv"
[1] "/Users/chronia/CHOP/GitHub/pbta-tumor-evolution/analyses/tumor-clone-inference/results/fastclone-input/PT_KTRJ8TFY.tsv"
[1] "PT_KTRJ8TFY"
[1] "/Users/chronia/CHOP/GitHub/pbta-tumor-evolution/analyses/tumor-clone-inference/results/pyclone-vi-input/PT_KTRJ8TFY.tsv"
[1] "/Users/chronia/CHOP/GitHub/pbta-tumor-evolution/analyses/tumor-clone-inference/results/fastclone-input/PT_KTRJ8TFY.tsv"
[1] "PT_KTRJ8TFY"
[1] "/Users/chronia/CHOP/GitHub/pbta-tumor-evolution/analyses/tumor-clone-inference/results/pyclone-vi-input/PT_KTRJ8TFY.tsv"
[1] "/Users/chronia/CHOP/GitHub/pbta-tumor-evolution/analyses/tumor-clone-inference/results/fastclone-input/PT_KTRJ8TFY.tsv"
[1] "PT_KTRJ8TFY"
[1] "/Users/chronia/CHOP/GitHub/pbta-tumor-evolution/analyses/tumor-clone-inference/results/pyclone-vi-input/PT_KTRJ8TFY.tsv"
[1] "/Users/chronia/CHOP/GitHub/pbta-tumor-evolution/analyses/tumor-clone-inference/results/fastclone-input/PT_KTRJ8TFY.tsv"
[1] "PT_KTRJ8TFY"
[1] "/Users/chronia/CHOP/GitHub/pbta-tumor-evolution/analyses/tumor-clone-inference/results/pyclone-vi-input/PT_KTRJ8TFY.tsv"
[1] "/Users/chronia/CHOP/GitHub/pbta-tumor-evolution/analyses/tumor-clone-inference/results/fastclone-input/PT_KTRJ8TFY.tsv"
[1] "PT_KZ56XHJT"
[1] "/Users/chronia/CHOP/GitHub/pbta-tumor-evolution/analyses/tumor-clone-inference/results/pyclone-vi-input/PT_KZ56XHJT.tsv"
[1] "/Users/chronia/CHOP/GitHub/pbta-tumor-evolution/analyses/tumor-clone-inference/results/fastclone-input/PT_KZ56XHJT.tsv"
[1] "PT_KZ56XHJT"
[1] "/Users/chronia/CHOP/GitHub/pbta-tumor-evolution/analyses/tumor-clone-inference/results/pyclone-vi-input/PT_KZ56XHJT.tsv"
[1] "/Users/chronia/CHOP/GitHub/pbta-tumor-evolution/analyses/tumor-clone-inference/results/fastclone-input/PT_KZ56XHJT.tsv"
[1] "PT_KZ56XHJT"
[1] "/Users/chronia/CHOP/GitHub/pbta-tumor-evolution/analyses/tumor-clone-inference/results/pyclone-vi-input/PT_KZ56XHJT.tsv"
[1] "/Users/chronia/CHOP/GitHub/pbta-tumor-evolution/analyses/tumor-clone-inference/results/fastclone-input/PT_KZ56XHJT.tsv"
[1] "PT_KZ56XHJT"
[1] "/Users/chronia/CHOP/GitHub/pbta-tumor-evolution/analyses/tumor-clone-inference/results/pyclone-vi-input/PT_KZ56XHJT.tsv"
[1] "/Users/chronia/CHOP/GitHub/pbta-tumor-evolution/analyses/tumor-clone-inference/results/fastclone-input/PT_KZ56XHJT.tsv"
[1] "PT_KZ56XHJT"
[1] "/Users/chronia/CHOP/GitHub/pbta-tumor-evolution/analyses/tumor-clone-inference/results/pyclone-vi-input/PT_KZ56XHJT.tsv"
[1] "/Users/chronia/CHOP/GitHub/pbta-tumor-evolution/analyses/tumor-clone-inference/results/fastclone-input/PT_KZ56XHJT.tsv"
[1] "PT_KZ56XHJT"
[1] "/Users/chronia/CHOP/GitHub/pbta-tumor-evolution/analyses/tumor-clone-inference/results/pyclone-vi-input/PT_KZ56XHJT.tsv"
[1] "/Users/chronia/CHOP/GitHub/pbta-tumor-evolution/analyses/tumor-clone-inference/results/fastclone-input/PT_KZ56XHJT.tsv"
[1] "PT_KZ56XHJT"
[1] "/Users/chronia/CHOP/GitHub/pbta-tumor-evolution/analyses/tumor-clone-inference/results/pyclone-vi-input/PT_KZ56XHJT.tsv"
[1] "/Users/chronia/CHOP/GitHub/pbta-tumor-evolution/analyses/tumor-clone-inference/results/fastclone-input/PT_KZ56XHJT.tsv"
[1] "PT_KZ56XHJT"
[1] "/Users/chronia/CHOP/GitHub/pbta-tumor-evolution/analyses/tumor-clone-inference/results/pyclone-vi-input/PT_KZ56XHJT.tsv"
[1] "/Users/chronia/CHOP/GitHub/pbta-tumor-evolution/analyses/tumor-clone-inference/results/fastclone-input/PT_KZ56XHJT.tsv"
[1] "PT_KZ56XHJT"
[1] "/Users/chronia/CHOP/GitHub/pbta-tumor-evolution/analyses/tumor-clone-inference/results/pyclone-vi-input/PT_KZ56XHJT.tsv"
[1] "/Users/chronia/CHOP/GitHub/pbta-tumor-evolution/analyses/tumor-clone-inference/results/fastclone-input/PT_KZ56XHJT.tsv"
[1] "PT_KZ56XHJT"
[1] "/Users/chronia/CHOP/GitHub/pbta-tumor-evolution/analyses/tumor-clone-inference/results/pyclone-vi-input/PT_KZ56XHJT.tsv"
[1] "/Users/chronia/CHOP/GitHub/pbta-tumor-evolution/analyses/tumor-clone-inference/results/fastclone-input/PT_KZ56XHJT.tsv"
[1] "PT_KZ56XHJT"
[1] "/Users/chronia/CHOP/GitHub/pbta-tumor-evolution/analyses/tumor-clone-inference/results/pyclone-vi-input/PT_KZ56XHJT.tsv"
[1] "/Users/chronia/CHOP/GitHub/pbta-tumor-evolution/analyses/tumor-clone-inference/results/fastclone-input/PT_KZ56XHJT.tsv"
[1] "PT_KZ56XHJT"
[1] "/Users/chronia/CHOP/GitHub/pbta-tumor-evolution/analyses/tumor-clone-inference/results/pyclone-vi-input/PT_KZ56XHJT.tsv"
[1] "/Users/chronia/CHOP/GitHub/pbta-tumor-evolution/analyses/tumor-clone-inference/results/fastclone-input/PT_KZ56XHJT.tsv"
[1] "PT_Z4BF2NSB"
[1] "/Users/chronia/CHOP/GitHub/pbta-tumor-evolution/analyses/tumor-clone-inference/results/pyclone-vi-input/PT_Z4BF2NSB.tsv"
[1] "/Users/chronia/CHOP/GitHub/pbta-tumor-evolution/analyses/tumor-clone-inference/results/fastclone-input/PT_Z4BF2NSB.tsv"
[1] "PT_Z4BF2NSB"
[1] "/Users/chronia/CHOP/GitHub/pbta-tumor-evolution/analyses/tumor-clone-inference/results/pyclone-vi-input/PT_Z4BF2NSB.tsv"
[1] "/Users/chronia/CHOP/GitHub/pbta-tumor-evolution/analyses/tumor-clone-inference/results/fastclone-input/PT_Z4BF2NSB.tsv"
[1] "PT_Z4BF2NSB"
[1] "/Users/chronia/CHOP/GitHub/pbta-tumor-evolution/analyses/tumor-clone-inference/results/pyclone-vi-input/PT_Z4BF2NSB.tsv"
[1] "/Users/chronia/CHOP/GitHub/pbta-tumor-evolution/analyses/tumor-clone-inference/results/fastclone-input/PT_Z4BF2NSB.tsv"
[1] "PT_Z4BF2NSB"
[1] "/Users/chronia/CHOP/GitHub/pbta-tumor-evolution/analyses/tumor-clone-inference/results/pyclone-vi-input/PT_Z4BF2NSB.tsv"
[1] "/Users/chronia/CHOP/GitHub/pbta-tumor-evolution/analyses/tumor-clone-inference/results/fastclone-input/PT_Z4BF2NSB.tsv"
[1] "PT_Z4BF2NSB"
[1] "/Users/chronia/CHOP/GitHub/pbta-tumor-evolution/analyses/tumor-clone-inference/results/pyclone-vi-input/PT_Z4BF2NSB.tsv"
[1] "/Users/chronia/CHOP/GitHub/pbta-tumor-evolution/analyses/tumor-clone-inference/results/fastclone-input/PT_Z4BF2NSB.tsv"
[1] "PT_Z4BF2NSB"
[1] "/Users/chronia/CHOP/GitHub/pbta-tumor-evolution/analyses/tumor-clone-inference/results/pyclone-vi-input/PT_Z4BF2NSB.tsv"
[1] "/Users/chronia/CHOP/GitHub/pbta-tumor-evolution/analyses/tumor-clone-inference/results/fastclone-input/PT_Z4BF2NSB.tsv"
[1] "PT_Z4BF2NSB"
[1] "/Users/chronia/CHOP/GitHub/pbta-tumor-evolution/analyses/tumor-clone-inference/results/pyclone-vi-input/PT_Z4BF2NSB.tsv"
[1] "/Users/chronia/CHOP/GitHub/pbta-tumor-evolution/analyses/tumor-clone-inference/results/fastclone-input/PT_Z4BF2NSB.tsv"
[1] "PT_Z4BF2NSB"
[1] "/Users/chronia/CHOP/GitHub/pbta-tumor-evolution/analyses/tumor-clone-inference/results/pyclone-vi-input/PT_Z4BF2NSB.tsv"
[1] "/Users/chronia/CHOP/GitHub/pbta-tumor-evolution/analyses/tumor-clone-inference/results/fastclone-input/PT_Z4BF2NSB.tsv"

Plot depth coverage

# Read color palette
palette_df <- readr::read_tsv(palette_file, guess_max = 100000, show_col_types = FALSE) %>% 
  mutate(tumor_descriptor = color_names)

# Define and order palette
palette <- palette_df$hex_codes
names(palette) <- palette_df$tumor_descriptor

# Define timepoints
timepoints = c("Diagnosis", "Progressive", "Recurrence", "Deceased", "Second Malignancy", "Unavailable")

for (i in 1:length(data_dir) ) { 
  
  # Create sample_name
  sample_name <-  unique(as.character(gsub(".call.cns", "", str_split_fixed(data_dir[i], "/", 13)[,11])))
  sample_name <- sort(sample_name, decreasing = FALSE)
  print(sample_name)
  
for (x in seq_along(sample_name) ) { 
  
  pyclone_input_subset <- pyclone_input %>%
    filter(Kids_First_Participant_ID == sample_name[x]) %>% 
    select(-c(Kids_First_Participant_ID)) #%>% 
   # mutate(tumor_descriptor = factor(tumor_descriptor),
    #     tumor_descriptor = fct_relevel(tumor_descriptor, timepoints)) %>% 
    #arrange(tumor_descriptor, sample_id)
  
  # Make this reproducible
  set.seed(2023)

  # Define label for plots
  Timepoint <- factor(x = pyclone_input_subset$tumor_descriptor, levels = timepoints)
  
  #######################
  # Create bxp ref_counts
    p <- print(ggplot(pyclone_input_subset, aes(sample_id, ref_counts, color = Timepoint)) + 
                 geom_jitter(width = 0.15, size = 0.7, alpha = 0.6) +
                 ggplot2::geom_boxplot(color = "black",
                              size = 0.25,
                              alpha = 0,
                              coef = 0) + # remove whiskers
                 theme_Publication() + 
                 scale_color_manual(values = palette,
                                    breaks = sort(names(palette))) +
                 #rotate() +
                 theme(axis.text.x = element_text(angle = 90)) +
                 stat_summary(fun.y=mean,shape=1,col='black',geom='point') +
                 labs(title = sample_name[x],
                      x = "sample_id",
                      y = "ref_counts",
                      color = "Timepoint"))
    
    # Save the plot
    ggsave(filename = paste0(sample_name[x], "-ref_counts.pdf"), 
         path = pyclone_plots_dir, 
         width = 6, 
         height = 5, 
         device = "pdf", 
        useDingbats = FALSE)
    

  #######################
  # Create bxp alt_counts
    p <- print(ggplot(pyclone_input_subset, aes(sample_id, alt_counts, color = Timepoint)) + 
                 geom_jitter(width = 0.15, size = 0.7, alpha = 0.6) +
                 ggplot2::geom_boxplot(color = "black",
                              size = 0.25,
                              alpha = 0,
                              coef = 0) + # remove whiskers
                 theme_Publication() + 
                 scale_color_manual(values = palette,
                                    breaks = sort(names(palette))) +
                 #rotate() +
                 theme(axis.text.x = element_text(angle = 90)) +
                 stat_summary(fun.y=mean,shape=1,col='black',geom='point') +
                 labs(title = sample_name[x],
                      x = "sample_id",
                      y = "alt_counts",
                      color = "Timepoint"))
    
    # Save the plot
    ggsave(filename = paste0(sample_name[x], "-alt_counts.pdf"), 
         path = pyclone_plots_dir, 
         width = 6, 
         height = 5, 
         device = "pdf", 
        useDingbats = FALSE)
  
  
}
}

[1] "PT_KTRJ8TFY"
[1] "PT_KTRJ8TFY"
[1] "PT_KTRJ8TFY"
[1] "PT_KTRJ8TFY"
[1] "PT_KTRJ8TFY"
[1] "PT_KTRJ8TFY"
[1] "PT_KTRJ8TFY"
[1] "PT_KTRJ8TFY"
[1] "PT_KZ56XHJT"
[1] "PT_KZ56XHJT"
[1] "PT_KZ56XHJT"
[1] "PT_KZ56XHJT"
[1] "PT_KZ56XHJT"
[1] "PT_KZ56XHJT"
[1] "PT_KZ56XHJT"
[1] "PT_KZ56XHJT"
[1] "PT_KZ56XHJT"
[1] "PT_KZ56XHJT"
[1] "PT_KZ56XHJT"
[1] "PT_KZ56XHJT"
[1] "PT_Z4BF2NSB"
[1] "PT_Z4BF2NSB"
[1] "PT_Z4BF2NSB"
[1] "PT_Z4BF2NSB"
[1] "PT_Z4BF2NSB"
[1] "PT_Z4BF2NSB"
[1] "PT_Z4BF2NSB"
[1] "PT_Z4BF2NSB"

Total number of mutations across timepoints and biospecimen sample per Patient case

for (i in 1:length(data_dir) ) { 
  
  # Create sample_name
  sample_name <-  unique(as.character(gsub(".call.cns", "", str_split_fixed(data_dir[i], "/", 13)[,11])))
  sample_name <- sort(sample_name, decreasing = FALSE)
  print(sample_name)
  
for (x in seq_along(sample_name) ) { 
  
  pyclone_input_subset <- pyclone_input %>%
    filter(Kids_First_Participant_ID == sample_name[x]) %>% 
    select(-c(Kids_First_Participant_ID))
  
  # Define parameters for function
  ylim = max(pyclone_input_subset$mutation_count)
  
  
   # Rename legend for timepoints
  Timepoint <- factor(pyclone_input_subset$tumor_descriptor)
  
  # Plot stacked barplot 
  print(ggplot(pyclone_input_subset, aes(x = sample_id, 
                                              y = mutation_count, 
                                              fill = Timepoint)) +  
               geom_col(position = position_stack(reverse = TRUE)) +
               geom_bar(stat = "identity", width = 0.5) + 
               scale_fill_manual(values = palette, breaks = sort(names(palette))) + 
               theme_Publication() + 
               theme(axis.text.x = element_text(angle = 85, 
                                                hjust = 1, 
                                                vjust = 1)) + 
               labs(title = paste(sample_name)) + 
               labs(x = "sample_id", y = "Total Mutations") +
               ylim(0, ylim)) 
  
  
}
}

[1] "PT_KTRJ8TFY"
[1] "PT_KTRJ8TFY"
[1] "PT_KTRJ8TFY"
[1] "PT_KTRJ8TFY"
[1] "PT_KTRJ8TFY"
[1] "PT_KTRJ8TFY"
[1] "PT_KTRJ8TFY"
[1] "PT_KTRJ8TFY"
[1] "PT_KZ56XHJT"
[1] "PT_KZ56XHJT"
[1] "PT_KZ56XHJT"
[1] "PT_KZ56XHJT"
[1] "PT_KZ56XHJT"
[1] "PT_KZ56XHJT"
[1] "PT_KZ56XHJT"
[1] "PT_KZ56XHJT"
[1] "PT_KZ56XHJT"
[1] "PT_KZ56XHJT"
[1] "PT_KZ56XHJT"
[1] "PT_KZ56XHJT"
[1] "PT_Z4BF2NSB"
[1] "PT_Z4BF2NSB"
[1] "PT_Z4BF2NSB"
[1] "PT_Z4BF2NSB"
[1] "PT_Z4BF2NSB"
[1] "PT_Z4BF2NSB"
[1] "PT_Z4BF2NSB"
[1] "PT_Z4BF2NSB"

sessionInfo()

R version 4.3.1 (2023-06-16)
Platform: x86_64-apple-darwin20 (64-bit)
Running under: macOS Ventura 13.6.1

Matrix products: default
BLAS:   /System/Library/Frameworks/Accelerate.framework/Versions/A/Frameworks/vecLib.framework/Versions/A/libBLAS.dylib 
LAPACK: /Library/Frameworks/R.framework/Versions/4.3-x86_64/Resources/lib/libRlapack.dylib;  LAPACK version 3.11.0

locale:
[1] en_US.UTF-8/en_US.UTF-8/en_US.UTF-8/C/en_US.UTF-8/en_US.UTF-8

time zone: America/New_York
tzcode source: internal

attached base packages:
[1] grid      stats     graphics  grDevices utils     datasets  methods   base     

other attached packages:
 [1] ggthemes_4.2.4  readxl_1.4.3    lubridate_1.9.3 forcats_1.0.0   stringr_1.5.1   dplyr_1.1.4     purrr_1.0.2     readr_2.1.4     tidyr_1.3.0    
[10] tibble_3.2.1    ggplot2_3.4.4   tidyverse_2.0.0

loaded via a namespace (and not attached):
 [1] utf8_1.2.4        generics_0.1.3    stringi_1.8.1     hms_1.1.3         digest_0.6.33     magrittr_2.0.3    evaluate_0.23     timechange_0.2.0 
 [9] fastmap_1.1.1     cellranger_1.1.0  rprojroot_2.0.4   fansi_1.0.5       scales_1.2.1      clonevol_0.99.11  textshaping_0.3.7 cli_3.6.1        
[17] rlang_1.1.2       crayon_1.5.2      bit64_4.0.5       munsell_0.5.0     withr_2.5.2       yaml_2.3.7        tools_4.3.1       parallel_4.3.1   
[25] tzdb_0.4.0        colorspace_2.1-0  vctrs_0.6.4       R6_2.5.1          lifecycle_1.0.4   bit_4.0.5         vroom_1.6.4       ragg_1.2.6       
[33] pkgconfig_2.0.3   pillar_1.9.0      gtable_0.3.4      glue_1.6.2        systemfonts_1.0.5 xfun_0.41         tidyselect_1.2.0  rstudioapi_0.15.0
[41] knitr_1.45        farver_2.1.1      htmltools_0.5.7   rmarkdown_2.25    labeling_0.4.3    compiler_4.3.1